Detecting Mannosidase Activities Using Ribonuclease B and Matrix-Assisted Laser Desorption/Ionization–Time of Flight Mass Spectrometry

Ribonuclease (RNase) B incubated with purified enzymes, whole bacterial cultures, or their separated components—cells and supernates—have been directly analyzed by matrix-assisted laser desorption/ionization–time of flight mass spectrometry (MALDI-ToF) to detect exomannosidases and to evaluate their...

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Bibliographic Details
Published in:Analytical biochemistry 2000-07, Vol.282 (2), p.165-172
Main Authors: Tarelli, Edward, Byers, Helen L., Wilson, Michael, Roberts, Gretta, Homer, Karen A., Beighton, David
Format: Article
Language:English
Subjects:
Amidohydrolases - analysis
Bacteria - enzymology
Culture Media
glycoforms
glycosidase
Hexosaminidases - analysis
MALDI-ToF mass spectrometry
Mannose - analysis
mannosidase
Mannosidases - analysis
Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase
oligomannose
Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
ribonuclease B
Ribonucleases - chemistry
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods
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Summary:Ribonuclease (RNase) B incubated with purified enzymes, whole bacterial cultures, or their separated components—cells and supernates—have been directly analyzed by matrix-assisted laser desorption/ionization–time of flight mass spectrometry (MALDI-ToF) to detect exomannosidases and to evaluate their specificities and location. Enzymatic cleavage was monitored by observing changes in RNase B glycoform population. Thus a nonspecific α-(1 → 2)-mannosidase activity converts the glycoprotein to its Man5 form, identifiable by its mass of 14,899 [M + H]+; this species subsequently is converted, by the actions of α-(1 → 3) and α-(1 → 6)-mannosidases, to the Man1 form via Man4, Man3, and Man2. The Man1 glycoform (which is readily isolated) has then similarly been used for identifying β-(1 → 4)-mannosidase and the derived Man0 form has served in turn as a natural substrate for β-(1 → 4) N-acetylglucosaminidase producing a species possessing a single asparagine-linked GlcNAc residue (mass 13,886). Mannose liberated from the actions of mannosidases can, if desired, be quantified by, for example, chromatography. The actions and specificities of endoglycosidases such as a peptide-N-glycosidase F (PNGase F) and of endo-N-acetlyglucosaminidases (e.g., endo-F and endo-H), which respectively cleave between the GlcNAcAsn and GlcNAcGlcNAc bonds of N-linked glycoproteins, are also demonstrable by MALDI-ToF analysis of RNase B (and derived products). From these digests the completely deglycosylated polypeptide corresponding to RNase A in which Asn has been converted to Asp (mass 13,684) and a species corresponding to RNase A + GlcNAc (mass 13,886) are produced, together with their corresponding free oligosaccharides which are amenable to analysis by both MALDI-ToF and by HPLC.
ISSN:0003-2697
1096-0309
DOI:10.1006/abio.2000.4606